Traumatic brain injury (TBI), or traumatic neuronal injury in vitro, causes neuronal apoptosis, in part through activation of caspases. Inhibition of caspase-3, in both in vivo or in vitro trauma models, reduces post-traumatic apoptosis, and improves functional outcomes in clinically relevant TBI models. However, some of these studies indicate that improvements often reflect only a delay in cell death, which still occurs eventually without the classical apoptotic phenotype. This suggests that caspase-independent pathways might play an important role in determining the final fate of cells. Recent work supports this hypothesis, demonstrating that caspase- independent apoptosis also contributes to neuronal cell death in a variety of in vitro model systems, and that translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus, in association with apoptotic morphological features, occurs after acute brain ischemia or TBI. Moreover, AIF translocation can occur under low energetic conditions, in association with activation of poly-ADP-ribose polymerase I (PARP-1) and reduction of nicotinamide adenine dinucleotide (NAD+). In contrast, caspase activation is generally associated with a more preserved bioenergetic state and requires adenosine 5'-triphosphate (ATP). Thus caspase-independent apoptosis may play a greater role than caspase-mediated cell death after a more severe injury, or within more central regions of the evolving lesion - sites at which cellular bioenergetic state is substantially compromised. AIF-mediated apoptosis may be initiated either by the same mechanisms responsible for intrinsic caspase activation or through PARP-1 activation. In the former, the role played by AIF becomes visible only when caspase activation has been blocked. In the latter, AIF is the main death-inducing factor. PARP-1 inhibition or PARP knockout animals, as well as knockout of the AIF carrier protein cyclophilin A, show reduced AIF translocation. We propose to utilize a well-established, controlled cortical impact (CCI) model of TBI in mouse, as well as selected in vitro models, to compare mechanisms underlying both caspase- dependent and caspase-independent programmed cell death of neurons and their relative roles as a function of injury severity and injury localization. Specific hypotheses include: 1) both caspase-independent and caspase-dependent pathways contribute to post-traumatic cell loss and associated neurological dysfunction after TBI, as well as to apoptotic neuronal cell death in cell culture models associated with DNA damage; 2) caspase-independent apoptosis is induced to a relatively greater degree than caspase-dependent cell death after more severe insults, or at more central regions of the expanding lesion, where bioenergetic state is reduced; 3) cell specific, inducible 'functional knockouts of AIF pro-death domains, as well as models in which AIF translocation is inhibited (PARP knockout, treatment with PARP inhibitors, or cyclophilin A knockout), show reduced apoptotic cell death after TBI or after cell injury in vitro, and; 4) inhibition of both caspase- dependent and caspase-independent cell death improves recovery after CCI in additive or synergistic fashion. We propose the following specific aims: 1) to compare the relative degree and location of caspase-dependent and caspase-independent neuronal cell death after mild, moderate or moderately-severe TBI; 2) to investigate the role of AIF in TBI-induced neuronal death and behavioral recovery by comparing two inducible, neuron-specific, pro-death domain selective AIF transgenic models versus their non-induced controls; 3) to evaluate the effects of cyclophilin A knockout on AIF translocation, apoptosis and behavioral outcome after TBI and in selected cell culture models and; 4) to evaluate the effects of two structurally-distinct PARP inhibitors or PARP-1 knockout on AIF translocation, apoptosis and behavioral outcome after TBI and in selected cell culture models, and determine whether such effects are additive or synergistic to that of caspase inhibition. PUBLIC HEALTH RELEVANCE: Traumatic brain injury (TBI) represents a major cause of death and disability in the United States. A better understanding of the mechanisms underlying TBI would offer the possibility of improving survival and insuring a more complete recovery.